Anti-inflammatory potential of aspergillus unguis SP51-EGY: TLR4-dependent effects & chemical diversity via Q-TOF LC-HRMS

Inflammation serves as an intricate defense mechanism for tissue repair. However, overactivation of TLR4-mediated inflammation by lipopolysaccharide (LPS) can lead to detrimental outcomes such as sepsis, acute lung injury, and chronic inflammation, often associated with cancer and autoimmune diseases. This study delves into the anti-inflammatory properties of “Aspergillus unguis isolate SP51-EGY” on LPS-stimulated RAW 264.7 macrophages. Through real-time qPCR, we assessed the expression levels of pivotal inflammatory genes, including iNOS, COX-2, TNF-α, and IL-6. Remarkably, our fungal extracts significantly diminished NO production and showed noteworthy reductions in the mRNA expression levels of the aforementioned genes. Furthermore, while Nrf2 is typically associated with modulating inflammatory responses, our findings indicate that the anti-inflammatory effects of our extracts are not Nrf2-dependent. Moreover, the chemical diversity of the potent extract (B Sh F) was elucidated using Q-TOF LC-HRMS, identifying 54 compounds, some of which played vital roles in suppressing inflammation. Most notably, compounds like granisetron, fenofibrate, and umbelliprenin were found to downregulate TNF-α, IL-1β, and IL-6 through the NF-κB signaling pathway. In conclusion, “Aspergillus unguis isolate SP51-EGY”, isolated from the Red Sea, Egypt, has been unveiled as a promising TLR4 inhibitor with significant anti-inflammatory potentials, presenting novel insights for their potential therapeutic use in inflammation. Supplementary Information The online version contains supplementary material available at 10.1186/s12896-024-00890-1.


Introduction
After injury or infection, an innate immune response arises in the body, leading to inflammation.Inflammation is considered a complex defense machinery to repair tissues in the body [1].The first line of defense to eradicate infections is macrophages.Toll-like receptors (TLRs) signaling pathways are activated by macrophages via lipopolysaccharide (LPS) with TLR4 ligates.This serves as an activating signal which leads to the initiation of intracellular pathways [2].The first inflammatory response involves the release of prostaglandins, histamine, and nitric oxide (NO) by inducible nitric oxide synthase (iNOS), which results in vasodilation, increased blood flow, and recruitment of leukocytes.Following activation of immune cells, pro-inflammatory cytokines like interleukin-6 (IL-6), interleukin-1 (IL-1), and tumor necrosis factor-alpha (TNF-α) escalate leukocyte permeability in the vascular regions by increasing leukocyte adhesion to endothelial cells.Homeostasis is necessary during inflammation as prolonged inflammation could lead to serious health issues [2,3].In addition, the nuclear factor erythroid 2-related factor 2 (Nrf2) is the primary redox homeostasis regulator.Nrf2 plays a crucial role in protecting cells from inflammation and oxidative stress by regulating the expression of phase II detoxification enzymes and oxidative stress response proteins, such as heme oxygenase-1 (HO-1), and oxidative stressinduced growth inhibitor 1 (OSGIN1) [4,5].
Natural products have been used since the beginning of time for healing applications.Currently, natural products are being used for the treatment of inflammatory diseases.Previously discovered herbal extracts have been shown to have key effects on TLR4 signaling pathways, a significant pathway for a pro-inflammatory response in the body [6].In addition, natural extracts have been shown to employ their anti-inflammatory effects by regulating the expression of pro-inflammatory cytokines in LPS-induced inflammatory response [7].
Marine-associated fungi have an excellent provenance for secondary metabolites, many of which have diverse biological activities and highly complicated structures, making them hard to provide economically via chemical synthesis [8].Furthermore, marine microorganisms can be cultured easily, which spreads high reproducibility and is a continual source of natural products [9].The Aspergillus genus contains more than 300 species that live in a variety of habitats, of which the marine-derived species produce variable structures of secondary metabolites, like alkaloids, phenolics, terpenoids, and peptides with prominent biological effects such as cytotoxic, antimicrobial, and anti-inflammatory activities [10].However, little is known about the chemical constituents and biological activities of "Aspergillus unguis isolate SP51-EGY" from the Red Sea, except that our previous work reported its antidiabetic effect [11].Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) is the dominant way to attain pure natural products for structure elucidation and evolution into therapeutic agents.In recent years, LC-HRMS use in natural product chemistry has grown in popularity, allowing for various analytical platforms for non-targeted, targeted, and suspect screening.LC-HRMS provides valuable structural input for detecting and exploring chemical substances identified from natural products [12].
This study aimed to find the highly effective antiinflammatory fungal extract and to identify its secondary metabolites using LC-HRMS, which plays an essential role in determining accurate masses and is used for comprehensive analysis in both positive and negative ionization modes to identify chemical compositions.This study proved that LC-HRMS is an efficacious and powerful analytical appliance for characterizing most of the identified compounds of "Aspergillus unguis isolate SP51-EGY" from Red Sea, Egypt.

Screening media and preparation of fungal extracts
For the fungus cultivation, four different broth media (A, B, C, and D) were used.The media were made up of the following components (g/L): Medium A (Sabouraud broth) contains dextrose (20 g/L) and peptone (10 g/L); Medium B (Nutrient broth) contains peptone (5 g/L), beef extract (1 g/L), yeast extract (2 g/L), and sodium chloride (6 g/L); Medium C (Potato dextrose broth) contains dextrose (20 g/L) and potato-infusion (200 g/L); Medium D (Malt extract broth) contains yeast extract (3 g/L) and malt extract (17 g/L).Secondary metabolite ethyl acetate extracts from static and shake conditions were abbreviated as: Sh F (shake filtrate) extract, Sh Cell (shake mycelia) extract, St F (static filtrate) extract, and St Cell (static mycelia) extract.

Determination of NO production using Griess method
RAW 264.7 cells were cultured in a 96-well plate for 2 h at a seeding density of 1 × 10 6 cells/mL.The cells were then stimulated with LPS (10 ng/mL) and co-treated with different fungal extracts at concentration of 10 µg/mL for 24 h.The NO production in the cell culture medium was determined using the Griess method through mixing 150 µL of the culture supernatant from each well with 130 µL of deionized water and 20 µL of Griess reagent and incubating for 30 min in the dark at room temperature as previously described [13].The absorbance was measured at 548 nm using SPECTROstar® Nano microplate reader (BMG LABTECH, Germany).NO concentration in each sample was eventually calculated using a NaNO 2 standard curve.

Determination of cytotoxicity using MTT assay
To confirm that the anti-inflammatory effect of the fungal extracts was not a result of cytotoxicity, MTT colorimetric assay was performed.On the 96-well plate containing cultured RAW 264.7 macrophages from the Griess experiment, MTT solution was added (1 mg/mL) for 2 h at 37 o C. MTT solution was then discarded and a volume of 100 µL of DMSO was added in order to solubilize the formed formazan crystals.The absorbance was measured at 540 nm using SPECTROstar® Nano microplate reader.Cell viability percentage was then determined relative to the control group.

Isolation of total RNA
RAW 264.7 cells were seeded at a density of 1 × 10 6 cells/mL in 6-well plates and incubated overnight.The cells were then stimulated with LPS (10 ng/mL) and cotreated with different fungal extracts at concentration of 10 µg/mL for 6 h.Total RNA was extracted from the cells using QiAzol lysis reagent.An amount of 1 µg of RNA from each sample was used to synthesize the cDNA using RevertAid First Strand cDNA Synthesis Kit according to manufacturer's protocol.cDNA was stored in -20 o C for further use in mRNA expression analysis.

Determination of inflammatory mRNA expression levels using quantitative real-time polymerase chain reaction (qPCR)
The qPCR was used for the determination of inflammatory mRNA expression levels.The cDNA was mixed with forward and reverse primers (Table 1), maxima SYBR green mix, and nuclease free water.The inflammatory genes selected for this experiment are: iNOS, COX-2, TNF-α, and IL-6.All mRNA expression levels were normalized to endogenous control (GAPDH) and the 2 −ΔΔCT method was used to evaluate the relative fold mRNA expression levels.The qPCR analysis was performed using ABI Prism 7500 system (Applied Biosystems) under the following conditions: initial holding stage at 95 °C for 10 min, followed by 40 PCR cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min.Primer design generation was done using NCBI Primer-Blast tool (Table 1).
Determination of mRNA expression levels of Nrf2driven ARE genes HO-1 and OSGIN1 in LPS-stimulated RAW 264.7 macrophages.
The qPCR primers for Nrf2-driven ARE Genes HO-1 and OSGIN1 were used to determine whether the antiinflammatory activity of the fungal extracts was associated with Nrf2 signaling pathway.The qPCR reactions were subjected to the same thermocycling conditions as the previous experiment.All mRNA expression levels were normalized to endogenous control (GAPDH) and the 2 −ΔΔCT method was used to evaluate the relative fold mRNA expression levels.
Quantification of the pro-inflammatory cytokine protein levels using enzyme-linked immunosorbent assay (ELISA) Protein expression levels of the pro-inflammatory cytokines, TNF-α and IL-6, were quantified by pre-coated ELISA plates (Elabscience®) according to the manufacturer's protocol.RAW 264.7 cells were seeded in 6-well plates (1 × 10 6 cells/mL) and incubated overnight.The cells were then stimulated with LPS (10 ng/mL) and cotreated with different fungal extracts at concentration of 10 µg/mL for 24 h.After incubation, the supernatants were collected, centrifuged at 1000× g for 20 min at 4 °C and then transferred to new microcentrifuge tubes to analyze the level of TNF-α and IL-6 proteins secreted into the cell culture medium.The supernatant was diluted in a ratio of 1:100 for TNF-α and 1:20 for IL-6 in buffered sample diluent.The absorbance was measured at 450 nm using SPECTROstar® Nano microplate reader.

Western blotting
Protein expression level of COX-2 was determined using Western blotting.RAW 264.7 cells were seeded in 6-well plates (1 × 10 6 cells/mL) and incubated overnight.The cells were then stimulated with LPS (10 ng/mL) and cotreated with different fungal extracts at concentration of 10 µg/mL for 24 h.Total proteins were extracted from the cells using cell lysis buffer containing a protease inhibitor cocktail, and the protein concentration was measured using Pierce™ BCA Protein Assay Kit.Western blotting was performed using a standard protocol.Briefly, proteins (10 µg) were separated by 10% SDS-PAGE and transferred to a PVDF membrane.After blocking with 5% non-fat dry milk in TBST for 1 h at room temperature, the membrane was incubated with primary antibodies; anti-COX-2 antibody (1:1000 dilution), and anti-GAPDH antibody (1:2500 dilution) overnight at 4 °C.After washing three times with TBST, the membrane was incubated with HRP-conjugated secondary antibody (1:15000 dilution) for 1 h at room temperature.Protein bands were visualized with Pierce™ ECL Western Blotting Substrate using ChemiDoc MP Imaging System (Bio-Rad Laboratories, CA, USA).

Q-TOF LC-HRMS spectroscopy analysis
For chromatographic separation, a 6530 Q-TOF LC/MS (Agilent Technologies) equipped with an autosampler (G7129A), a Quat.Pump (G7104C), and a Column Comp (G7116A) were used at the Faculty of Pharmacy, Fayoum University.The injection volume was set at 5 L. The analytes were separated on an Agilent Technologies Zorbax RP-18 column (dimensions: 150 mm 3 mm, dp = 2.7 m) at a flow rate of 0.3mL/min.ESI was used to obtain mass spectra in (+) and (-) ionization modes with a capillary voltage of 4500 V.The mass spectra were recorded in the 50-3000 m/z range.The gas temperature and drying gas flow were 200 O C and 8 L/min, respectively.The skimmer and fragmentator voltages were set at 65 and 130 V, respectively, and collision energy was 10 V.The nebulization pressure was 58psig.Elution was with Solvent A (Water 0.1%formic acid) and Solvent B (Acetonitrile 0.1%formic a), the flow rate was 0.3mL/min.Gradient elution started with 2% B, reaches 10% B at 15 min and 20% B at 35 min, 50% B at 60 min, 70% B at 80 min and then 100% B at 100 min.

Statistical analysis
Statistical significance between groups was determined using one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls post hoc test, where P-value < 0.05 was considered statistically significant.The data from three independent experiments are presented as the mean ± standard error of the mean (SE).Comparative analysis between experimental groups was performed using SigmaPlot (Version 14.0; Systat Software, Chicago, IL, USA).

Determination of NO production in LPS-stimulated RAW 264.7 macrophages
Macrophages play a key role in the innate immune response [14,15].LPS, a major component in the cell wall of gram-negative bacteria can induce the production of inflammatory mediators, and is frequently used to evaluate the anti-inflammatory effects of drugs [16].The production of inflammatory mediators including NO is increased in LPS-stimulated macrophages.NO is a bioactive signaling molecule which has a key role in modulating the inflammatory response [17].Therefore, the anti-inflammatory effect of the fungal extracts was evaluated using LPS-stimulated RAW 264.7 macrophages.The NO production in the cell culture medium was determined using the Griess method and according to the measured NaNO 2 standard curve.It was shown that NO production was significantly increased in the cell culture medium of LPS-stimulated macrophages (19.8 µM) compared to the control (11.2 µM).
As for the extracts in medium (A-D), NO production was significantly reduced in all samples in medium A and C in comparison to LPS-stimulated cells.As for medium B, only Sh F and St Cell extracts significantly reduced NO production, resulting in levels of 12.9 µM and 14.4 µM, respectively, in comparison to LPS-stimulated cells, and for medium D, Sh F and Sh Cell extracts significantly reduced NO production, resulting in levels of 7.2 µM and 6.8 µM, respectively, in comparison to LPS-stimulated cells (Fig. 1).These results validate that some of the fungal extracts have significant effect on reducing NO production in LPS-stimulated RAW 264.7 cells and highlight the potential anti-inflammatory effect on RAW 264.7 cells [18].
To ensure that the observed NO reduction was not due to a reduction in cell viability, MTT assay was used to measure the cytotoxicity of RAW 264.7 macrophages after treatment with the extracts (medium A-D).Our results showed that no cytotoxicity was observed at the tested concentrations on the cultured cells.As a result, the reduction in NO production observed in the LPSstimulated cells after treatment with the extracts was not due to cytotoxicity.The anti-inflammatory effect of these extracts was further experimented on to assess their antiinflammatory effect using the same concentration (10 µg/ mL) of NO production (Fig. 2).

Determination of mRNA expression levels of the proinflammatory markers iNOS, COX-2, TNFα, and IL-6 in LPSstimulated RAW 264.7 macrophages
During inflammation, the immune system triggers the production of pro-inflammatory cytokines (TNF-α, IL-6), as well as enzymes (iNOS, COX-2) [19].Current research is emphasizing the exploration of bioactive compounds with the capacity to suppress the production of these inflammatory mediators.Natural products are gaining attention as potent and safe anti-inflammatory agents, owing to their capability to modulate gene expression of diverse inflammatory mediators, thus offering promising avenues for therapeutic intervention [20].Hence, to investigate the anti-inflammatory activities of the fungal extracts, the mRNA expression level of iNOS, COX-2, TNF-α, and IL-6 was determined using qPCR (Fig. 3).Overall, eight samples from the different extraction media, with the highest anti-inflammatory action as evidenced by Griess assay, were selected for further anti-inflammatory studies.The results from qPCR further confirmed our previous results obtained from the Griess analysis.For LPS-stimulated macrophages, significant upregulation was observed for iNOS, COX-2, TNFα, and IL-6 mRNA levels compared to the control.After treatment with the fungal extracts, the mRNA expression levels were significantly reduced.For the iNOS mRNA expression levels, significant reduction was observed for Sh F and St Cell in medium B, and Sh Cell in medium C (Fig. 3A).As for COX-2 mRNA expression levels, all samples in medium (A-D) showed a highly significant reduction in COX-2 expression in comparison to LPSstimulated macrophages (Fig. 3B).Furthermore, the following samples showed significant reduction in TNF-α mRNA expression levels: Sh F in medium A, Sh F and St Cell in medium B, Sh Cell in medium C, and Sh F in medium D. However, St F (medium A), St F (medium C) and Sh Cell (medium D) did not show any significant reduction in TNF-α mRNA expression levels (Fig. 3C).Finally, for IL-6 mRNA expression levels, all 8 samples in all extraction medium (medium A-D) showed a highly significant reduction in IL-6 mRNA expression levels in comparison to LPS-stimulated macrophages (Fig. 3D).Our findings are in agreement with the results reported by Anh et al. indicating that nitrogen-containing secondary metabolites from Aspergillus unguis showed antiinflammatory activity by suppressing the production of NO and the expression of iNOS and IL-6 in LPS-stimulated RAW 264.7 macrophages [21].In a previous study conducted by Cao et al., it has also been demonstrated that sterols isolated from Aspergillus unguis inhibited the

Determination of mRNA expression levels of Nrf2-driven ARE genes HO-1 and OSGIN1 in LPS-stimulated RAW 264.7 macrophages
The Nrf2-antioxidant response element (ARE) signaling pathway, which is recognized as a key regulatory system maintaining the intracellular redox homeostasis, also has a role in reducing inflammation [4].The Nrf2-ARE activity can be attributed to modulating the expression of antioxidant and detoxifying enzymes, such as HO-1 and OSGIN1 [23,24].Additionally, recent studies demonstrated a correlation between the expression of inflammatory mediators, the NF-κB pathway, macrophage metabolism, and the Nrf2/ARE system [25].To examine whether the inhibitory effect of the selected fungal extracts on LPS-mediated inflammation in RAW 264.7 macrophages was related to activation of the Nrf2driven genes, the mRNA expression levels of HO-1 and OSGIN1 was analyzed by qPCR.Our data showed no significant upregulation in HO-1 and OSGIN1 mRNA expression between control, LPS-stimulated cells, and treated groups in mediums A-D.This is an indication that both HO-1 and OSGIN1 are not affected by any of the treatments (Fig. 4).These results propose that the anti-inflammatory effects of the fungal extracts are Nrf2-independent.

Determination of protein levels of the pro-inflammatory cytokines TNFα and IL-6 in LPSstimulated RAW 264.7 macrophages
Cytokines are key signaling proteins, which are produced to regulate the interactions between different cell types involved in the immune response [26].Cytokines are mainly produced by phagocytic cells and natural killer (NK) cells during innate immune responses, while they are mostly secreted by lymphocytes and antigen-presenting cells (APCs) during adaptive immune responses.Therefore, cytokines coordinate the crosstalk between the innate and adaptive immune systems.Among these, TNF-α and IL-6 are the major pro-inflammatory cytokines involved in the inflammatory response [27,28].In the present study, the protein expression levels of the pro-inflammatory cytokines TNF-α and IL-6, were assessed by ELISA.This was to confirm that the anti-inflammatory responses observed on the mRNA level carried through to the protein level.A concentration of 10 ng/mL of LPS significantly increased the proinflammatory response of RAW 264.7 macrophages as indicated by the elevated protein levels of the assessed cytokines (Fig. 5).However, at concentration of 10 µg/ mL of the selected fungal extracts (Sh F and St Cell in medium B and Sh Cell in medium C), significant reduction was observed in protein levels of the pro-inflammatory cytokines TNFα and IL-6 in LPSstimulated RAW 264.7 macrophages.

Determination of protein expression of COX-2 using western blotting
The iNOS induction in relation to COX-2 has been formerly reported in previous studies.In this context, it was shown that selective COX-2 inhibitors reduce COX-2-mediated generation of prostacyclin (PGI2).Moreover, PGI2 was demonstrated to induce iNOS expression, resulting in the production of NO.Thus, it is likely that the fungal extracts might have reduced NO production via a selective inhibition of COX-2 [29].A concentration of 10 ng/mL of LPS significantly increased the proinflammatory response of RAW 264.7 macrophages as indicated by the elevated protein levels of COX-2 (Fig. 6).However, at concentration of 10 µg/mL of the selected fungal extracts (Sh F and St Cell in medium B and Sh Cell in medium C), significant reduction was observed in COX-2 protein levels in LPSstimulated RAW 264.7 macrophages.

Q-TOF LC-HRMS spectroscopy analysis
LC-MS coupled with QTOF is a cutting-edge analytical technique for identifying unknown bioactive compounds in natural product extracts.The key to obtaining pure natural products for structure elucidation and development into therapeutic agents is liquid chromatographyhigh-resolution mass spectrometry (LC-HRMS).
Based on the results of medium B extract (Sh F) represented by its significant anti-inflammatory effects, prompted us to investigate its chemical composition with Q-TOF LC-HRMS spectroscopy analysis.In our study, the chemical composition of a highly bioactive extract (Sh F) from medium B was elucidated using Q-TOF LC-HRMS analysis.All compounds were identified depending on their m/z value from MS spectra in both positive and negative ionization modes ([M + H] + /[M-H] − , Fig. 7), using an Agilent LC/MS Mass Hunter Qualitative Software for preliminary identification, then confirmed from many other libraries databases (Figs. 8 and 9).In positive and negative ionization modes, Q-TOF LC-HRMS identified a total of 33 and 21 compounds, respectively (Table 2A, 2B).

Q-TOF LC-HRMS analysis [positive ionization mode
According to our findings, the positive ionization mode was primarily characterized by the identified ions of peptides, fatty acids, amides, triglycerides, and others.

Structural fragmentation study of peptides
For isoleucyl-alanine and L-isoleucyl-L-proline; the complementary ions produced by the cleavage of the amino-alkyl group's carbonyl C-C bond result in the characteristic fragment ion protonated "2-methylbutan-1-amine" with 86 m/z (C 5 H 12 N), Fig. 8).
The MS/MS spectrum in positive ionization mode shows three distinct pathways for fragmentation.The data from the MS 3 spectrum of Angiotensin IV could further confirm this conclusion (Figs.10A and 9A, Table 2A).On the other hand, Angiotensin IV   2A, 2B).
Bioactive peptides have been shown to have antiinflammatory effect in macrophages by inhibiting the NO/iNOS and PGE2/COX-2 pathways while suppressing the production of pro-inflammatory cytokines such IL-1β and TNF-α [30].When THP-1 cells were stimulated with TNF-α, Cysteine and Histidine reduced NF-κB activity [31].It was confirmed that the polypeptide "Angiotensin IV" repressed inflammation in the brains of rats with chronic cerebral hypoperfusion (CCH), as it significantly reduced the levels of TNF-α, IL-1β, IL-6 and IL-12 in the brains of rats with CCH (CCH) [32].
In-vitro, saturated fatty acids (SFAs) were found to directly stimulate inflammatory gene expression via TLR4 signaling.The relative potency of various (SFAs) varied with chain length, with lauric acid (12:0) having the highest activity, whereas myristic acid (14:0) and stearic acid (18:0) having surprisingly little pro-inflammatory activity.Unlike SFAs, monounsaturated-fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) did not activate TLR4 signaling.These researchers were able to demonstrate that pretreatment of cells for 3 h with a     variety of PUFAs or oleic acid (18:1) significantly reduced the subsequent pro-inflammatory effect of lauric acid treatment.They went on to demonstrate that PUFAs' ability to inhibit inflammatory responses induced by LPS or lauric acid was dependent on TLR4 [33].Valproic acid (VPA) inhibits the CD45 high F4/80 low macrophage subset as well as the production of pro-inflammatory cytokines/ chemokines such as CXCL1, IL-5, IL-6, and IL-10.(VPA) specifically decreased tissue NF-кB2 p100 protein [34].
The identified constituents were found at m/z 281.2736Fatty acid amides, played critical roles in inflammation suppression [35].Oleamide suppressed LPS-induced iNOS and COX-2 mRNA in RAW 264.7 cells, as well as inflammatory cytokines; TNF-α, IL-1β and IL-6 [36].Anandamide inhibited NO and IL-6 release by LPSstimulated J774 macrophages, as well as IL-12 and IL-23 production and increased IL-10 production by activated microglia via JNK and ERK1/2 activation and NF-κB [37].

Other compounds
In addition; ten other compounds were identified, three of which were previously reported to have anti-inflammatory effect; Hydroxy-ibuprofen, at m/z 222.1267  2A).
Hydroxy Ibuprofen is an Ibuprofen metabolite.Ibuprofen is an anti-inflammatory agent with IC50s of 13 M and 370 M for COX-1 and COX-2, respectively [38].Resveratrol was a potent inhibitor of the NO and cytokine release in activated macrophages and microglia [39].Rifamycin-O also antagonized TNF-α and LPS-induced NF-κB activities and inhibited IL1β-induced synthesis of inflammatory chemokine, IL8 [40].

Halogenated, phenolic compounds and glycosides
Furthermore, eleven compounds were identified in negative ionization mode, from which the followings : Granisetron, Rt (63.2B), were previously reported to have anti-inflammatory activity.
The amide "Granisetron" significantly reduced TNF-a, IL-6, HMGB1, and NF-kB.It also reduced the expression of the receptor for advanced glycation end, TLR4, in liver tissue, as well as pyroptosis, by decreasing NLRP3, IL-1β, and caspase-1 [43].Fenofibrate decreases the expression and secretion of TNF-α, IL-1β, and IL-6 via the NF-κB signaling pathway, making them therapeutic targets for attenuating inflammation in hepatic pathological progression [44].
Umbelliprenin (UMB), a natural sesquiterpene coumarin, may reduce inflammation by lowering IL-17 levels in the blood.UMB exerts its anti-inflammatory effects by modulating distinct cytokine release/inhibition types [45].Alpha-CEHC is a vitamin E derivative that reduces LPS-induced gene expression of TNF-α, IL-1β, IL-6, and iNOS.While high concentrations increased gene expression in peritoneal macrophages [46].Studies in rat models have indicated that vitamin E exhibits antiinflammatory properties by suppressing IL-1 and IL-6.Vitamin E has also been shown to inhibit COX-2, the enzyme involved in inflammatory reactions [47].Piceid is a Resveratrol glycoside: Resveratrol inhibited NO and cytokine release in activated macrophages and microglia [39].

Conclusions
The intricate interplay of TLR4 in immune responses underscores its dual role as both protector and potential provocateur.Malfunctioning TLR4 signaling can inadvertently amplify immune responses, thereby inducing conditions like sepsis, acute lung injury, and pathological chronic inflammation, often linked to cancer and autoimmune maladies.Central to this inflammatory response is the production of NO by iNOS, which triggers the subsequent activation TNF-α, and IL-6.Notably, this study delved into the potential Nrf2-dependency of the anti-inflammatory properties of "Aspergillus unguis isolate SP51-EGY" extracts, revealing their effects to be independent of Nrf2 modulation.A significant modulation in the expression of inflammatory markers, such as iNOS, COX-2, TNF-α, and IL-6 was observed using realtime qPCR.The extract labeled (B Sh F) was particularly potent in this regard.Further chemical profiling of this extract via Q-TOF LC-HRMS unveiled a rich tapestry of 54 bioactive compounds, with several playing pivotal roles in inflammation suppression.Key compounds such as granisetron, fenofibrate, and umbelliprenin were shown to attenuate inflammatory pathways, particularly the NF-κB signaling cascade.Marine-associated fungi, like the "Aspergillus unguis isolate SP51-EGY" from the Red Sea, have emerged as bounteous reservoirs of secondary metabolites, displaying diverse biological activities and intricate structures.This study not only underscores the potential of LC-HRMS as a formidable analytical tool but also highlights the pressing need for further research into the bioaccessibility, bioavailability, and toxicological profiles of these fungal extracts.Future endeavors should also encompass animal model studies, paving the way for the potential therapeutic commercialization of these promising extracts.

Fig. 1
Fig. 1 Measurement of NO production of extracts found in medium (A-D) on RAW 264.7 macrophages.After treatment of LPS-stimulated RAW 264.7 macrophages (1 × 10 6 cells/mL) with 10 µg/mL of extracts in medium (A-D) and cultured overnight.NO production in the cell culture medium was measured using the Griess method.LPS-stimulated cells only showed high significance in comparison to control.All extracts in medium A and medium C showed a significant decrease in NO production in comparison to LPS-stimulated cells.However, Sh Cell and St F extracts in medium B and St F and St Cell in medium D did not show any significant decrease in comparison to LPS-stimulated cells.Results are presented as the mean ± SE (n = 3).Statistical significance was calculated by one-way ANOVA followed by Student-Newman-Keuls post-hoc test.$ P < 0.05 vs. control cells.* P < 0.05 vs. LPS-stimulated cells

Fig. 2
Fig. 2 Measurement of cytotoxicity on RAW 264.7 macrophages using MTT assay.After treatment of LPS-stimulated cells with 10 µg/mL of extracts found in medium (A-D) and cultured overnight, cell viability was determined using MTT assay.All extracts in medium (A-D) did not show any cytotoxicity on RAW 264.7 cells.Results are presented as the mean ± SE (n = 3).Statistical significance was calculated by one-way ANOVA followed by Student-Newman-Keuls post-hoc test.$ P < 0.05 vs. control cells.* P < 0.05 vs. LPS-stimulated cells

Fig. 5
Fig. 5 Determination of protein levels of the pro-inflammatory cytokines TNF-α and IL-6 in the cell culture medium of the LPS-stimulated RAW 264.7 macrophages using ELISA.The selected fungal extracts (Sh F and St Cell in medium B and Sh Cell in medium C) showed a significant reduction of the LPS-stimulated upregulation of the pro-inflammatory cytokines TNF-α (A) and IL-6 (B).Results are presented as the mean ± SE (n = 3).Statistical significance was calculated by one-way ANOVA followed by Student-Newman-Keuls post-hoc test.$ P < 0.05 vs. control cells.* P < 0.05 vs. LPS-stimulated cells

Table 2
Characterization of secondary metabolites from "A.Unguis isolate SP51-EGY" fungal extract by Q-TOF LC-HRMS analysis (A = positive, B = negative ionization mode)